The present invention relates to nucleic acids comprising a nucleotide sequence encoding at least a portion of an enzyme which catalyzes the synthesis of chitin in arthropods, inhibitors directed to said enzyme, and a method for developing said inhibitors.
Chitin is a carbohydrate homopolymer of xcex2(1xe2x86x924) linked N-acetylglucosamine. Chitin is a structural polysaccharide occurring mainly in the cell wall of some fungi and in exoskeleton of the arthropods.
Chitin is synthesized by the action of specialized enzymes, referred to as chitin synthases, that catalyze the polymerization of N-acetylglucosaminyl residues into chitin from uridine 5xe2x80x2-diphospho-N-acetylglucosamine.
Chitin synthases belong to a group of enzymes that catalyze the synthesis of linear structural polysaccharides and are collectively termed as processive glycosyltransferases. The primary structures of the known processive glycosyl transferases (i.e. the fungal chitin synthases, the prokaryotic and eukaryotic cellulose synthases, the prokaryotic and eukaryotic hyaluronan synthases, the prokaryotic alginate syntheses and the rhizobial chito-oligosaccharide syntheses) present some sequence similarities suggesting that these UDP-sugar utilizing enzymes show a similar mode of action. Despite the similarities, the various processive glucosyltransferases display substantial differences mainly in their substrate specificities, their activation and inhibition parameters, the size and conformation of their products.
Since several arthropod species are considered as plant and animal pests, the inhibition of the chitin synthesis machinery has been viewed in particular as an attractive target for the development of pest control agent.
Known insect chitin synthesis inhibitors, either natural or synthetic, fall within two main categories: (a) direct inhibitors of chitin synthesis such as the Streptomyces antibiotics Nikkomycins and Polyoxins and (b) insect growth regulators that interfere indirectly but drastically with the synthesis of chitin in insects, such us the benzoylphenyl-urea derivatives.
Although the studies for the identification of these compounds have a history of several decades, the action of chitin synthesis inhibitors is still inadequately understood. This is due to both the limited availability of suitable insect chitin synthase enzyme preparations and to the lack of any molecular insight on the chitin synthesis and its regulation in insects.
Thus, the technical problem underlying the present invention is to provide new inhibitors for the chitin synthesis in order to prevent damages caused by arthropods.
The solution to the above technical problem is achieved by providing the embodiments characterized in the claims.
In particular, there is provided a nucleic acid which comprises a nucleotide sequence encoding at least a portion of an enzyme which catalyzes the synthesis of chitin in arthropods.
The terms xe2x80x9cnucleic acidxe2x80x9d and xe2x80x9cnucleotide sequencexe2x80x9d refer to endogenously expressed, semisynthetic, synthetic or chemically modified acid molecules of deoxyribonucleotides and/or ribonucleotides. In a preferred embodiment of the present invention the nucleotide sequence include sequences as illustrated in FIGS. 13 (SEQ ID NO:1), allelic derivatives of said sequences and DNA-sequences degenerated as a result of the genetic code for said sequences. It also includes DNA sequences hybridizing under stringent conditions with the nucleotide sequence defined above. Although said allelic, degenerate and hybridising sequences may have structural divergences due to (naturally occurring) mutations, such as small deletions or substitutions, they will usually still exhibit essentially the same useful properties, allowing their use in basically the same applications.
In a preferred embodiment the present invention relates to an isolated DNA sequence encoding a portion of an enzyme which catalyzes the synthesis of chitin in arthropods. Specific embodiments include DNA sequences which are characterized by the ability to hybridize to the DNA sequence represented in FIG. 1 at e.g. 55xc2x0 C. or encode a polypeptide reactive with an antibody against the polypeptide containing the deduced amino acid sequence in FIGS. 1 to 3. The invention also relates to a DNA expression construct containing the isolated DNA sequences and encoding an enzyme having the specificity described above.
The present invention also relates to recombinant molecules comprising the nucleic acid as described above optionally linked to an expression-control sequence. Such vectors may be useful in the production of at least said portion of an enzyme in stable or transiently transformed cells. Several animal, plant, fungal and bacterial systems may be used for the transformation and subsequent cultivation process. Preferably, expression vectors which can be used in the invention contain sequences necessary for the replication in the host cell and are autonomously replicable. It is also preferable to use vectors containing selectable marker genes which can be easily selected for transformed cells. The necessary preparation is well known to those skilled in the art.
It is another object of the invention to provide a host cell transformed by an expression plasmid of the invention and capable of producing at least said portion of an enzyme. Examples of suitable host cells include various eukaryotic and prokaryotic cells, such as E. coli, insect cells, plant cells, mammalian cells, and fungi such as yeast.
Another object of the present invention is to provide a polypeptide comprising at least a portion of an enzyme which catalyzes the synthesis of chitin in arthropods. The term xe2x80x9cpolypeptidexe2x80x9d includes the complete enzyme or a biologically active or inactive fragment thereof encoded by the sequences described above and displaying preferably biologically features of said enzyme. The amino acid sequences of specially preferred polypeptides contain the sequences displayed in FIGS. 13 (SEQ ID NO:2).
It is a further aspect of the invention to provide a process for the production of the above mentioned polypeptide. Such a process comprises cultivating a host cell being transformed with a nucleic acid sequence of the present invention in a suitable culture medium and purifying the produced polypeptide. Thus, this process allows the production of the sufficient amount of the desired polypeptide for use in applications described herein below. The host cell is obtainable from bacteria such as E. coli, from fungi, such as yeast, from plants such as tobacco, potato, or Arabidopsis, and from animals, in particular vertebrate cells such as the CHO-cells.
The invention also relates to the methods for assaying the effects of various compounds on the expression and the biological activity of arthropod chitin synthases. In these methods the polypeptide in enzymatically active form, preferably the enzyme chitin synthase of an arthropod is produced by recombinant DNA techniques in which an isolated DNA sequence encoding the enzyme is expressed from a DNA expression construct as outlined above.